Changes in phosphofructokinase activity upon exposure of muscles and muscle extracts to injurious agents

A study has been made on changes of outflux, extractability and activity of phosphofructokinase (PPK) under the action of heating, and of urea on the frog's skeletal muscles and on their extracts. Under the action of heating on muscles, the decrease of PPK activity (to 35%) is first revealed 34--36 degress C, when decrease of excitability and the contracture are not yet detected, and the extractability of the total water-soluble protein does not change. At the start of contracture, and at the decrease of excitability (at 38 degrees C) PPK in the muscle loses its activity. The thermolability of PPK is the greatest one compared to all the enzymes investigated before. The data on the high thermolability of PPK are discussed in terms of the regulating role of PPK in glycolysis. The PPK activity of extracts of muscles altered by urea, during the action not accompanied by the death of muscles, does not change. At the irreversible disappearance of muscle excitability, PPK is inactivated. PPK in the cell is more stable to injuring agents than PPK in isolated state.     

Changes in hexokinase activity during muscle alteration

Changes in extractability and activity of hexokinase (HK) were studied under the action of heating and of urea on skeletal muscles of Rana temporaria L., and besides the stability of this enzyme in muscle extract to those agents in vitro was examined. Under a 15 minutes heating of muscle, a decrease in extractability (the activity calculated for 1 g of tissue) and activity (the activity calculated for 1 mg of protein) of hexokinase is first revealed at 37 degrees C. Then the enzyme extractability decreases gradually in accordance to the decrease in extractability of the total water-soluble protein; the level of hexokinase activity attained at 37 degrees does not change up to 40 degrees. At 42 degrees the activity of the enzyme is completely inhibited. Under the heating of the muscle extract, the decrease of enzyme activity takes place at 36 degrees, the level achieved being stable up to 42 degrees C. Under the action of urea on the muscle at the reversible phase of alteration (1 M urea from 5 minutes to 2 hours at room temperature, 1 M urea for 9 hours at + 4 degrees C), hexokinase activity increases, calculated for 1 g of tissue and for 1 mg of protein. Under the irreversible disappearance of muscle excitability (1 M urea during 9 hours, 2 M urea during 2 hours at room temperature) no hexokinase activity was revealed. The activation of the enzyme is discussed in connection with the data on the increase of ATP content in muscle under the urea alteration. The treatment of the enzyme in muscle extract with 1 M urea decreases its activity in 30 minutes down to 67%; the level achieved does not change during 20 hours.     

The nature of changes in the activity of water-soluble enzymes during the action of injurious agents on the muscles. V. A study of the extractability of lactate dehydrogenase from intact and altered muscles

A study was made of lactate dehydrogenase (LDH) extractability from intact and thermally injured muscles of Rana temporaria L. in the 0.15 M KCl solution and in the non-electrolyte medium. A 15 minute incubation of intact muscles and those treated with heat at 38, 42, 44 and 46 degrees C in the 0.15 M KCl solution led to a much higher extraction of LDH than in the non-electrolyte medium. Following heating at 38 degrees C, causing irreversible injury of muscles, the LDH extractability in the non-electrolyte medium is seen to fall, whereas in the 0.15 M KCl solution it remained at the same level as after LDH extraction from intact muscles. The decrease in LDH extractability in non-electrolyte medium from thermally injured muscles may be due to the increase in LDH binding with thermolabile structural components of muscle. One of the components of muscle cell, known to bind LDH, is actomyosin, since it is isolated from muscles together with LDH, which cannot be removed by actomyosin reprecipitation or by increasing the volume of washing solution.     

Changes in rat testis glandulocytes under deep hypothermia]

White mongrel rats weighing 180-220 g were daily cooled in water at 4-5 degrees C up to 21-23 degrees C in the rectum. The animals were killed on the 1, 5, 10, 15, 20 and 25th days of experiment. The total amount of glandulocytes counted per a cross section of the tubule did not change throughout the experiment. The total weight of pictures of 100 glandulocytes (taken randomly), obtained through a drawing apparatus on a standard paper was reliably decreased by 28%. By the end of the observation period the amount of degenerating glandulocytes increased 2 times and that of slightly active in a functional respect so-called "small" glandulocytes increased 4 times, while the number of active ("large") glandulocytes diminished approximately 1,5 times. The activity of SDG and ICDG in interstitial cells dropped by 26% and 18%. The activity of LDG was 52% greater. The G-6-PhDG activity was 40% greater while the activity of clue enzyme steroidgenase--3beta-OSD under hypothermia was inhibited by 36%.     

Effect of prolactin on the enzymatic activity of thyroid follicular cells during stress due to surgery and subsequent subcutaneous inflammation

In order to reveal prolactin (Prl) effect on the enzymatic activity of the thyroid follicular cells at a subcutaneous inflammation, 2 series of experiments have been performed on 100 non-inbred male rats with body mass of 150-170 g. In order to produce the inflammation, under ether narcosis celloidin globules are introduced subcutaneously in the abdominal wall. The test animals are given 0.125 mg of "Lactin" (the Soviet preparation of bovine Prl) in 0.5 ml of isotonic solution once every day, beginning immediately after the operation. At the same time the control animals are given 0.5 ml of isotonic solution. The time of observations are--2, 12 h, 1, 2, 3, 5, 10, 15 days after the operation. As a result of quantitative cytochemical investigation of the enzymatic activity in the thyroid follicular cell, multiphasic dynamics of SDG, LDG, G-6-PhDG, NADH-DG, NADPhH-DG, MAO activity and changes in carbohydrate metabolism type at various time of the subcutaneous inflammation are revealed. Daily injections of Prl against the background of the inflammation reduce amplitude of fluctuations of the enzymatic activity in thyrocytes. At late stages of inflammation (up to 2 days) Prl slows down the decrease of SDG, G-6-PhDG, NADH-DG, NADPhH-DG activity and accelerates decrease of LDH activity. At early stages of inflammation (3-15) Prl slows down decrease of NADPhH-DG, NADH-DG activity and decreases LDH and NADH-DG activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative histoenzymologic characteristics of the human submandibular salivary glands]

Quantitative activity of oxidative-reductive and hydrolytic enzymes obtained during operation was investigated in different parts of the human submandibular salivary glands. Quantitative estimation of enzymic activity was done by photometry of the negatives prepared on MY-phi-6. Comparative examination of enzymatic activity made it possible to state that according to the peculiarities of metabolic processes, the cells of striated and intralobular secreting ducts are similar to the cells of the secreting terminal parts. A high activity of NADP-diaphorase, G-6-PhDG and acid phosphatase in the epithelium of the secreting ducts, and parallelism, stated between histoenzymatic and morphologic criteria of functional activity proved the participation of these structural-functional units in secret-producing processes. A high activity of NAK-diaphorase, LDG and acid phosphatase in all the parts and in the secreting ducts system, in particular, ensures, besides the processes of protein secret formation, an active transport of natrium and potassium, formation of final saliva and its discharge.     

Effects of inhibitors on chloride outflux from cerebrospinal fluid

Movement of chloride from cerebrospinal fluid (CSF) to brain or blood is one of the factors that may be involved in regulation of CSF [Cl-], which is important to CSF acid-base balance. We made quantitative measurements of the unidirectional outflux of radiolabeled chloride (38Cl, half-life 37.3 min) from CSF in anesthetized dogs, using ventriculocisternal perfusion (VCP). The outflux of 38Cl from CSF was determined from the difference between the movements of 38Cl and dextran using a one-compartment model. VCP was performed at a rate of 1.4 ml/min for 14 min, and then slowed to 0.28 ml/min. The 38Cl activity decreased to a steady-state level approximately 12% lower than that of dextran within 40-50 min. Under control conditions for the first run (n = 24), the flux was 0.042 +/- 0.003 (SE) ml/min. The outflux under control conditions (n = 6) tended to increase over three separate determinations in a 6-h period, being 136 +/- 19% of the first run on the second run, and 143 +/- 24% on the third. There were no significant changes in 38Cl outflux compared with control ratios after the inclusion of bumetanide in the VCP fluid (n = 6), which inhibits sodium-coupled Cl- transport, with acetazolamide (n = 6), which inhibits carbonic anhydrase, or with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (n = 6), an inhibitor of carrier-mediated anion exchange. These results suggest that the outward movement of chloride from CSF occurs mostly by passive diffusion and is not by mediated transport.     

Serum enzymatic activity in oncological patients treated with carminomycin]

Karminomycin effect on the activity of some serum enzymes, such as hexokinase (HK), lactate dehydrogenase (LDG), its isoenzymes and glucose-6-phosphatase (G-6-P-ase) was studied. Biochemical assays were applied to 52 patients with neglected malignant tumors. The course dose of the drug was on the average 72mg. The objective antitumor effect was registered in 15 patients. A reliable increase in the values of LDG-5 and G-6-P-ase was observed after the treatment course in the combined group consisting of all the patients subjected to the biochemical assay. Normalization of the serum enzyme spectrum was observed in 15 patients effectively treated with karminomycin: activity of HK and the cathode fractions of LDG decreased. When treatment with karminomycin was ineffective (37 cases), the changes in the enzymatic activity recorded before the treatment further aggraviated. It was found that the level of G-6-P-ase in the patients' treated with karminomycin increased independent of the treatment effect which was probably associated with its toxic effect on the liver. The increase was reversible.     

Contracture of the myocardial fibers of the frog ventricle after high frequency stimulation while the calcium channels were blocked by manganese ions]

The contractile force of the myocardial strip of the frog ventricle stimulated by impulses of 0.5 Hz was diminished to 3--5% of the initial value when perfused with Ringer's solution containing 2.5 mM manganese. Under this condition the action potential duration was significantly decreased. An increase in frequency of stimulation up to 5 Hz leads to the development of contracture. The amplitude of contracture was about 30% of the initial contractile force in normal perfusion solution. The amplitude of contracture was more than doubled under the effect of ouabain (2 X X 10(-6) g/ml). Similar experiments with lanthanum failed to discover contracture produced by the increase of the stimulation frequency. In these experiments ouabain was also ineffective. It is supposed that contracture observed in the presence of manganese was caused by nonelectrogenic calcium transport into the muscle fibers.     

Effects of diltiazem and tetrodotoxin on the development of hypoxic contracture of the guinea pig myocardium

In the isolated electrically stimulated right ventricular papillary muscles the onset of hypoxic contracture occurred 7 +/- 1.2 min and reached maximum 29.2 +/- 4.6 min after the onset of hypoxia. Switching off of the stimulation and diltiazem (10(-6) M) or tetrodotoxin (3 X 10(-6) M) administration delayed the development of the hypoxic contracture and decreased its maximum level. The protective action of diltiazem was noted only in the presence of rhythmical stimulation. It was concluded that, in addition to the influx of Ca ions through calcium channels, the influx of Na ions through sodium channels was important in the development of hypoxic contracture.     

Effect of training on skeletal muscle injury from downhill running in rats

Experiments were conducted to test the hypothesis that injury to skeletal muscle in rats resulting from prolonged downhill running is prevented to a greater extent by prior downhill training than by either uphill or level training. Changes in plasma creatine phosphokinase (CPK) activity and glucose-6-phosphate dehydrogenase (G-6-PDase) activity in the soleus (S), vastus intermedius (VI), and medial head of triceps brachii (TM) muscles were evaluated as markers of muscle injury 48 h after 90 min of intermittent downhill running (16 m . min -1). Prior to this acute downhill run, groups of rats were trained by either downhill (-16 degrees), level (0 degrees), or uphill (+16 degrees) running (16 m . min -1) for 30 min/day. Training duration was either 5 days or 1 day. A training effect (i.e., reduced muscle injury) was indicated if muscle G-6-PDase or plasma CPK activity in a trained group following the 90-min downhill run was not different from that of nonexercised control animals and/or if it was lower than that of nontrained runners. A significant training effect was achieved in all three muscles with 5 days of either downhill or level training, but only in S after 5 days of uphill training. Elevation of plasma CPK activity was prevented by 5 days of training on all three inclines.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of temperature acclimation on NADP-dependent dehydrogenase activity in the carp liver and various mechanisms of its regulation

Acclimation of carp both to the temperature fall (from 20 to 5 degrees C) and rise (from 20 to 30 degrees C) induces an increase in activity of cytoplasmic liver NADPH-generating enzymes--glucose-6-phosphate dehydrogenase (G6PDG) and malic-enzyme (ME) 6-phosphogluconate dehydrogenase (6PGDG) and NADP-isocitrate dehydrogenase (NADP-IDG) activities are unchanged. Actinomycin D does not prevent cold activation of G6PDG but blocks activation of ME. "Warm" G6PDG has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for glucose-6-phosphate and "warm" ME has minimal Km value for malate at 25 degrees C "Cold" G6PDG and ME have the warmest Km values at 5 degrees C. Isozyme composition of cytoplasmic G6PDG (2 bands with Rf 0.16 and 0.20) does not change within the limits of 5-30 degrees C. The prolactin action on G6PDG and ME is similar to the effect of cold acclimation (activity increases Km value decreases, isozyme pattern (for G6PDG) remains unchanged). It is supposed that activation of G6PDG and ME during cold adaptation may be a result of the prolactin action on substrate-binding ability without changes in the enzyme biosynthesis and isozyme pattern.     

The effect of exposure to 60Co accelerated electrons and gamma quanta on the activity of oxidative and hydrolytic enzymes in the rat brain]

In experiments with 112 male Wistar rats it was shown that accelerated electrons (85 Gy) caused a significant increase in activities of succinate dehydrogenase (SDG) by 15.8% and lactate dehydrogenase (LDG) by 17.0%, and a decrease in activities of alkaline phosphatase (AP) and monoamine oxidase (MAO) by 10.6 and 7.8% respectively within the sensorimotor region of the cerebral cortex immediately after irradiation. Activity of SDG and MAO decreased (by 16.4% and 7.8% respectively) in the caudate nucleus over the same period of time. An increase in the accelerated electron dose from 85 to 500 Gy did not change the direction and the rate of the radiation response of the enzymes. Exposure of rats to 60Co gamma quanta (75 Gy) increased SDG and LDG activity (by 21.4 and 17.3% respectively) within the sensorimotor cortex as late as 10 min after irradiation. A repeated significant increase in SDG and LDG activity was observed 2 hr after irradiation.     

Post-mortem glycolysis in ox skeletal muscle. Effect of temperature on the concentrations of glycolytic intermediates and cofactors

1. Post-mortem changes in the concentrations of the following compounds in ox sternomandibularis muscles stored in nitrogen at 1 degrees , 5 degrees and 15 degrees are reported: P(i) creatine phosphate, hexose monophosphates, fructose diphosphate, triose phosphates, alpha-glycerophosphate, phosphoglycerates, lactate, ATP, ADP, AMP, NAD(+) and total nucleotides. Some results obtained with muscles stored at 37 degrees are included. 2. At the time the muscles were placed at controlled temperatures (about 1.5hr. post mortem) the phosphorus in the compounds measured accounted for 91+/-6% (s.d.) of the total acid-soluble phosphate. 3. The results indicated that at all temperatures the activities of the phosphorylase and phosphofructokinase steps limited the rate and the extent of post-mortem glycolysis. 4. The large variations in hexose monophosphate concentrations during storage indicated that the ratio of phosphorylase to phosphofructokinase activity varied considerably with time and temperature. 5. Between 3.5 and 7hr. post mortem the rates of glycolysis and of ATP turnover were not slower at 5 degrees that at 15 degrees , and were probably faster at 1 degrees . The significance of this finding is discussed.     

Eccentric exercise-induced injury to rat skeletal muscle

These experiments were designed to study skeletal muscle pathology resulting from eccentric-biased exercise in rats. The effects on the muscles of running on a treadmill on a 0 degrees incline (similar amounts of concentric and eccentric contractions), down a 16 degrees incline (primarily eccentric contractions), and up a 16 degrees incline (primarily concentric contractions) at 16 m . min-1 for 90 min were assessed by following postexercise changes in 1) plasma creatine kinase and lactate dehydrogenase activities, 2) glucose-6-phosphate dehydrogenase (G-6-PDase) activity (bio- and histochemically) in the physiological extensor muscles, and 3) histological appearance of the muscles. The data indicate the following. 1) Whereas all exercise protocols resulted in elevations of plasma enzymes immediately after running, only eccentric exercise caused late phase elevations 1.5-2 days postexercise. 2) Significant increases in muscle G-6-PDase activity, which were always associated with accumulations of mononuclear cells, always occurred within some muscles of each extensor group 1-3 days following downhill and uphill running and did not occur following level running; the increases in activity were usually of lower magnitude in the muscles of uphill runners than in those of downhill runners; the deeply located, predominantly slow-twitch muscles were most affected by both down- and uphill running. 3) Muscle histology demonstrated localized disruption of normal banding patterns of some fibers immediately after exercise and accumulations of macrophages in the interstitium and in some (less than 5%) muscle fibers by 24 h postexercise in the deep slow muscles of the antigravity groups. Although the data generally indicated that eccentric exercise causes greater injury to the muscles, questions remain.     

The nature of the changes in the activity of water-soluble enzymes in action on the muscles of damaging agents. VI. Glyceraldehyde-3-phosphate dehydrogenase binding and enzyme adsorption by F-actin in ghost muscle fiber]

A possibility of binding glyceraldehyde-3-phosphate dehydrogenase (GAPhDG) in frog (Rana temporaria L.) skeletal muscles was studied by measuring its solubilization in 0.15 M KCl and by its presence in isolated actomyosin. Using a 0.15 M KCl solution, more GAPhDG was extracted from intact muscles and muscles treated with heat at 38, 42 and 46 degrees C for 15 min than in a non-electrolyte medium. Actomyosin isolated from muscles reveals GAPhDG activity which cannot be removed by actomyosin reprecipitation. In myosin-, troponin- and tropomyosin-free single glycerinated muscle fibres (ghost fibres) GAPhDG absorption to F-actin was shown. It is suggested that under thermal injure of muscle cells, the increase in GAPhDG binding with thermolabile proteins of actomyosin complex may occur.     

Sites of action of a peptide neurohormone that controls hindgut muscle activity in the cockroach Leucophaea maderae

A peptide neurohormone from the brain and nervous system of the Madeira cockroach Leucophaea maderae has stimulating effects on both the mechanical and electrical events of hindgut visceral muscle. The peptide initiated action potentials at silent recording sites in the circular muscles of the rectum after prior treatment with tetrodotoxin (10⁻⁶ g/ml). The neurohormone also caused an increase in the amplitude and frequency of spontaneous postsynaptic potentials. However, the isolated hindgut failed to respond to the neurohormone after depolarization in high potassium saline solutions. Both the potassium contracture and the action of the neurohormone were calcium dependent.Although some hindguts were responsive to the neurohormone in a Ca free medium, such preparations failed to respond in 0·5 mM EGTA. Moreover, 1 mM Mn blocked the action of the peptide. The sodium ion was also essential for effective hormone action. These results suggest the presence of a loosely bound source of Ca at the surface of muscle membranes that in some way interacts with the neurohormone to change muscle excitability.     

Insulin secretion in patients deficient in glucose-6-phosphate dehydrogenase

Intravenous (IVGTT) and oral glucose tolerance tests (OGTT) were carried out in 12 men with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and in 11 normal men. The race, the mean age and body mass index were similar in the G-6-PD deficient and in the normal men. No significant differences were demonstrated between mean plasma glucose levels in the G-6-PD deficient subjects and those in the normal men during IVGTT and OGTT. In contrast the insulin levels were significantly lower for the G-6-PD deficient subjects as compared to the controls at 30 minutes (P less than 0.04) in the OGTT and at 1 min (P less than 0.001), 3 min (P less than 0.001), 5 min (P less than 0.001) and 10 minutes (P less than 0.002) in the IVGTT. All indexes of first phase insulin release were also significantly (P less than 0.001) lower in G-6-PD deficient men. These results emphasize the metabolic importance of G-6-PD in the process of glucose induced insulin release.     

Metabolism and evaporative heat loss in the dik-dik antelope (Rhynchotragus kirki)

1. Under controlled conditions, the rate of oxygen consumption (VO2) respiratory frequency, evaporative water loss, heat balance, rectal (Trec) and surface temperatures were determined in the dik-dik antelopes at ambient temperatures (Ta) ranging from 1 to 44 degrees C. 2. The thermal neutral zone was found to be between 24 and 35 degrees C. 3. Respiratory frequency ranged between 27 and 630 breaths/min. 4. At a Ta of 44 degrees C, 95% of the heat produced by the dik-dik was lost via respiratory evaporation. Despite an increase in Trec, cutaneous evaporation did not increase. 5. During panting, VO2 increased in accordance with the expected Q10 effect, contrary to earlier findings. 6. Measurements of circadian rhythm [LD 12:12 (7-19) CT26 degrees C] in VO2 showed that the minimum VO2 (0.42 ml O2/g/hr) occurred at midnight while the maximum (0.78 ml O2/g/hr) occurred at midday. The 24 hr mean VO2 was 0.61 ml O2/g/hr. 7. These measurements suggest that in nature, determinants other than light may be responsible for triggering the variations observed in VO2.     

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.